Selective AMPK activator leads to unfolded protein response downregulation and induces breast cancer cell death and autophagy

Abstract

Aims
AMPK plays a critical role regulating cell metabolism, growth and survival. Interfering with this enzyme activity has been extensively studied as putative mechanism for cancer therapy. The present work aims to identify a specific AMPK activator for cancer cells among a series of novel heterocyclic compounds.

Materials and methods
A series of novel hybrid heterocyclic compounds, namely naphtoquinone-4-oxoquinoline and isoquinoline-5,8-quinone-4-oxoquinoline derivatives, were synthesized via Michael reaction and their structures confirmed by spectral data: infrared; ¹H and ¹³C NMR spectroscopy (COSY, HSQC, HMBC); and high-resolution mass spectrometry (HRMS). The novel compounds were screened and tested for antitumoral activity and have part of their mechanism of action scrutinized.

Key findings
Here, we identified a selective AMPK activator among the new hybrid heterocyclic compounds. This new compound presents selective cytotoxicity on breast cancer cells but not on non-cancer counterparts. We identified that by specifically activating AMPK in cancer cells, the drug downregulates unfolded protein response pathway, as well as inhibits mTOR signaling.

Significance
These effects, that are selective for cancer cells, lead to activation of autophagy and, ultimately, to cancer cells death. Taken together, our data support the promising anticancer activity of this novel compound which is a strong modulator of metabolism.

Full text

Gene annotation of nuclear receptor superfamily genes in
the kissing bug Rhodnius prolixus and the effects of
20-hydroxyecdysone on lipid metabolism
P. V. P. Nascimento*, F. Almeida-Oliveira†,
A. Macedo-Silva*, P. Ausina†, C. Motinha*,
M. Sola-Penna*†and D. Majerowicz*‡
*Departamento de Biotecnologia Farmacêutica,
Faculdade de Farmácia, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; †Instituto de Bioquímica
Médica Leopoldo de Meis, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil; and ‡Instituto Nacional
de Ciência e Tecnologia em Entomologia Molecular, Rio
de Janeiro, Brazil
Abstract
The hormone 20-hydroxyecdysone is fundamental for
regulating moulting and metamorphosis in immature
insects, and it plays a role in physiological regulation
in adult insects. This hormone acts by binding and acti-
vating a receptor, the ecdysone receptor, which is part
of the nuclear receptor gene superfamily. Here, we ana-
lyse the genome of the kissing bug Rhodnius prolixus
to annotate the nuclear receptor superfamily genes.
The R. prolixus genome displays a possible duplica-
tion of the HNF4 gene. All the analysed insect organs
express most nuclear receptor genes as shown by
RT-PCR. The quantitative PCR analysis showed that
the RpEcR and RpUSP genes are highly expressed in
the testis, while the RpHNF4-1 and RpHNF4-2 genes
are more active in the fat body and ovaries and in the
anterior midgut, respectively. Feeding does not induce
detectable changes in the expression of these genes in
the fat body. However, the expression of the RpHNF4-2
gene is always higher than that of RpHNF4-1. Treating
adult females with 20-hydroxyecdysone increased the
amount of triacylglycerol stored in the fat bodies by
increasing their lipogenic capacity. These results indi-
cate that 20-hydroxyecdysone acts on the lipid metab-
olism of adult insects, although the underlying
mechanism is not clear.
Keywords: nuclear receptor, genome, gene expres-
sion, triacylglycerol, fatty acid synthesis.
Introduction
The steroid ecdysone is the primary regulator of the ecdy-
sis and metamorphosis processes in immature insects
(Yamanaka et al., 2013). At this stage of development,
the prothoracic gland produces ecdysone (Huang
et al., 2008) and then releases it into the haemolymph.
The target tissues are responsible for metabolizing ecdy-
sone into 20-hydroxyecdysone (20-HE), the active hor-
mone (Petryk et al., 2003). In adult insects, ovarian
follicles are significant sites of ecdysone production
(Hagedorn et al., 1975; Cardinal-Aucoin et al., 2013;
Swevers, 2019), and this hormone regulates various pro-
cesses such as ovogenesis (Swevers, 2019), circadian
rhythm (Uryu et al., 2015) and programmed cell death
(Nicolson et al., 2015).
20-HE acts through a signalling cascade that induces the
expression of a series of early and late genes, and it is
mediated by its receptor, a heterodimer formed by the pro-
teins ecdysone receptor (EcR) and ultraspiracle (USP)
(Yao et al., 1992; Hill et al., 2013), both of which belong to
the nuclear receptor superfamily (Fahrbach et al., 2012).
Nuclear receptors are transcription factors that are usually
regulated directly by hydrophobic ligands capable of cross-
ing the plasma membrane (Bain et al., 2007), and they reg-
ulate lipid, glucose and energy metabolism and the
detoxification of xenobiotics in a coordinated manner
(Chiang, 2005). The proteins in this superfamily have a
highly conserved structure among the different taxa. A typ-
ical nuclear receptor basically consists of an N-terminal
region with little conservation; a DNA-binding domain
(DBD); a ligand-binding domain (LBD); and a region that
connects the two domains, which is called hinge (Bain
Correspondence: D. Majerowicz, Av. Carlos Chagas Filho, 373, CCS, Bloco
B-1
andar - Sala 35, Cidade Universitária, Rio de Janeiro, RJ, Brazil,
21941-902. majerowicz@pharma.ufrj.br
We dedicate this manuscript to Professor Franklin David Rumjanek for ded-
icating his life to Brazilian science.
© 2021 The Royal Entomological Society 1
Insect Molecular Biology (2021) doi: 10.1111/imb.12696

et al., 2007). The proteins from the NR0 family are an
exception and have no LBD (Perl et al., 2013). The DBD
recognizes and docks on the response elements within
the gene promoters. The LBD has a variety of functions
(Bain et al., 2007). It contains the ligand’s binding pocket
and has a ligand-regulated transcriptional activation func-
tion, which interacts with different coactivators as needed
(Xu and Li, 2003). Furthermore, this domain is involved in
the nuclear receptor dimerization process, which is essen-
tial for a high-affinity DNA interaction (Kumar and
Chambon, 1988).
From an evolutionary perspective, the nuclear receptor
superfamily is highly conserved among the Insecta class,
with few species showing duplications or gene absences
(Fahrbach et al., 2012). The genome of the fruit flies Dro-
sophila melanogaster and Drosophila pseudoobscura
codify 18 typical nuclear receptors and three other genes
from the NR0 family (King-Jones and Thummel, 2005;
Clark et al., 2007). In the mosquitoes Anopheles gambiae
and Aedes aegypti, a new gene, called NR2E6, was found
(Holt et al., 2002; Cruz et al., 2009). Similarly, the red flour
beetle Tribolium castaneum, the honey bee Apis mellifera
and the parasitoid wasp Nasonia vitripennis all have 19 typ-
ical nuclear receptor genes in their genomes (Velarde
et al., 2006; Bonneton et al., 2008; Werren et al., 2010).
Conversely, the genome of the silkworm Bombyx mori
has lost the HR83 gene orthologue, and the pea aphid
Acyrthosiphon pisum does not contain the HR96 and
NR2E6 genes (Cheng et al., 2008; Christiaens et al., 2010).
The nuclear receptors’functions in metamorphosis and
ecdysis have been known for a long time. For example,
E75,E78,HR3,EcR,USP,HR78,FTZ-F1 and HR4 partic-
ipate in the signalizing cascade for the steroid hormone
ecdysone in D. melanogaster (Sullivan and
Thummel, 2003). However, some studies have uncovered
the involvement of these proteins in the energy metabo-
lism, reproduction, lifespan and behaviour of adult insects
(Schwedes and Carney, 2012).
Ecdysone also plays a role in lipid metabolism
(Lehmann, 2018), but the effects appear to vary by spe-
cies, stage of life and test organ. Treating B. mori larvae
with 20-HE increases the lipolytic activity in the fat body
as measured by the increased haemolymph lipid levels,
lipase activity and Brummer lipase expression in the organ,
and there is a consequent reduction in the amount of tria-
cylglycerol (TAG) in the fat body. However, 20-HE has no
direct effect on the fat body but acts by reducing food con-
sumption, which causes fasting and lipid mobilization
(Wang et al., 2010). However, 20-HE has the opposite
effect on D. melanogaster adult females; hormone treat-
ment increases the TAG levels due to increased food
intake (Sieber and Spradling, 2015). In adult A. aegypti
females, EcR knockdown by RNA interference (RNAi)
increases the amount of TAG and the lipid droplet size in
the fat body, possibly due to the reduction in the β-oxidation
capacity caused by a decrease in hepatocyte nuclear fac-
tor 4 (HNF4) expression, a central regulator of lipid catabo-
lism (Palanker et al., 2009; Hou et al., 2015; Wang
et al., 2017). EcR knockdown in D. melanogaster larvae
results in similar effects (Kamoshida et al., 2012). How-
ever, in adult D. melanogaster females, the use of condi-
tional EcR mutants reduces TAG accumulation in the
oocytes and fat body due to reduced sterol-responsive
element-binding protein (SREBP) activation and expres-
sion, and consequently, there is decreased expression of
various lipid anabolic genes, including the lipophorin recep-
tor (Sieber and Spradling, 2015).
The kissing bug Rhodnius prolixus is an essential vector
of Chagas disease in Latin America (de Fuentes-Vicente
et al., 2018) and a traditional model for the study of metab-
olism and physiology (Nunes-da-Fonseca et al., 2017).
Even Sir Wigglesworth used this insect in his classic stud-
ies on ecdysis endocrinology between 1930 and 1940
(Wigglesworth, 1934, 1940). However, information on the
synthesis and functions of 20-HE in R. prolixus is still
scarce. In nymphs, the prothoracic gland appears to pro-
duce ecdysone in a feeding-dependent manner; the hor-
mone levels rise 6 days after feeding and peak after
13 days (Beaulaton et al., 1984). In adult insects, the ova-
ries begin ecdysone production just 2 h after feeding, and
the hormone reaches its maximum levels after 4 days.
However, the amount of circulating ecdysone varies
according to the circadian rhythm (Cardinal-Aucoin
et al., 2013). 20-HE has several roles in R. prolixus, includ-
ing neuropeptide production (Wulff et al., 2018), Trypano-
soma cruzi development (Gonzalez et al., 1999; Cortez
et al., 2002, 2012), immune system modulation
(Azambuja et al., 1997; Figueiredo et al., 2006; Genta
et al., 2010) and structural changes in the gut (Garcia
et al., 1998; Gonzalez et al., 1999; Albuquerque-Cunha
et al., 2004).
Although R. prolixus is extensively used to model lipid
metabolism (Gondim et al., 2018), the role of 20-HE, if
any, has never been explored in this respect. Here, we ana-
lyse the R. prolixus genome (Mesquita et al., 2015) to
locate and annotate the nuclear receptor superfamily
genes. We measured the expression of the identified
genes by reverse transcriptase-PCR (RT-PCR) and quan-
titative PCR (qPCR). Finally, we explored the effects of
20-HE on TAG accumulation in the R. prolixus fat body.
Results
The R. prolixus genome codifies 22 genes from the nuclear
receptor superfamily (Table 1). There are two genes
(RPRC003216 and RPRC009166) from the NR0 family
that contain only a DBD. The DBD from both genes has a
high shared identity relative to the DBDs in proteins from
2 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

other insects. The NR1 family is made up of six genes in
R. prolixus (RPRC000824,RPRC00853,RPRC001794,
RPRC009045,RPRC009064 and RPRC14174). The
DBD and LBD showed shared identities ranging from
99% to 52% and 85% to 44%, respectively, compared to
other nuclear receptors from the same family. The largest
family is the NR2, with nine genes (RPRC000767,
RPRC001064,RPRC002557,RPRC006737,
RPRC007025,RPRC008212,RPRC009755,
RPRC010625 and RPRC013330). The DBD and LBD
were also highly conserved. The RpERR (oestrogen-
related receptor, ERR) gene is the only one within the
NR3 family. This gene does not have a VectorBase code
because it was not present at the preliminary annotation.
RpERR was found after a secondary search using the con-
sensus ERR DBD as a query and the TblastN algorithm
against the R. prolixus genome contigs. The GeneWise
algorithm was used to predict the gene sequence. How-
ever, unfortunately, the LBD could not be completely found,
and the cloning and sequencing of its full complementary
DNA sequence would be necessary to obtain the entire
gene sequence. The DBD is complete and has approxi-
mately 84–88% shared identity when compared to other
insects. RPRC001680 is the only gene representing the
NR4 family, and it showed high conservation. The NR5
family consists of two genes (RPRC001915 and
RPRC002968), and the shared identity of DBD and LBD
was above 75%. Finally, the gene RPRC012796 is the only
one belonging to the NR6 family, and it also showed a high
degree of conservation on the DBD and LBD. Two full den-
drograms constructed with the DBD and LBD sequences of
the nuclear receptor from R. prolixus,D. melanogaster,
T. castaneum,B. mori,A. pisum,A. aegypti,A. mellifera
and D. pulex are shown in supplementary Figs S1 and
S2. We used these results to define the gene orthology
between the species and to name the R. prolixus genes
(Table 1).
R. prolixus encodes two genes from the NR0 family
(Table 1). Fig. S3 shows a dendrogram constructed using
the DBD from the NR0 proteins of different species. As
shown, the generated tree had little statistical support,
probably due to the low number of species used here.
We used the DBD and LBD sequences of proteins within
the NR1 family to generate a relationship tree (Fig. 1(A),
(B)). Both dendrograms showed good clade separation
and statistical support. The genes E75,E78,HR3,EcR
and HR96 constitute individual and monophyletic clades.
Based on these results, it is possible to say that
R. prolixus has one-to-one orthologous genes for E75,
HR3,EcR and HR96. Furthermore, we were able to identify
Table 1.;R. prolixus nuclear receptor superfamily genes
Identity (%)
D. melanogaster
T.
castaneum B. mori A. pisum A. aegypti A. mellifera
Gene
Nuclear
Receptor
(NR)
nomenclature
Rhodnius
accession
DNA-
binding
domain
(DBD)
ligand-
binding
domain
(LBD) DBD LBD DBD LBD DBD LBD DBD LBD DBD LBD
RpKnrl-1 NR0A3 RPRC003216 89 a 96 a b a 98 a b a 92 a
RpKnrl-2 NR0A3 RPRC009166 88 a 95 a b a 99 a b a 93 a
RpE75 NR1D3 RPRC000853 97 c 52 c 97 c 91 c 97 c 97 c
RpE78-1 NR1E1 RPRC009045 c 55 c 67 c 44 c 66 c 55 c 64
RpE78-2 NR1E1 RPRC009064 c c c c c c c c c c c c
RpHR3 NR1F4 RPRC000824 96 59 97 82 95 55 97 65 96 60 97 82
RpEcR NR1H1 RPRC014174 88 66 98 85 88 62 96 68 88 67 99 80
RpHR96 NR1J1 RPRC001794 72 48 68 49 d 49 b b 71 54 71 56
RpHNF4-1 NR2A4 RPRC001064 87 61 86 66 87 63 83 62 84 58 84 d
RpHNF4-2 NR2A4 RPRC008212 88 69 88 75 88 73 88 71 86 63 85 d
RpUSP NR2B4 RPRC013330 90 41 94 68 92 43 91 66 94 44 94 70
RpHR78 NR2D1 RPRC006737 82 36 89 53 88 31 90 49 84 28 92 52
RpTLL NR2E2 RPRC007025 83 38 83 44 81 42 82 33 82 40 88 50
RpHR51 NR2E3 RPRC002557 99 68 93 75 d 65 99 78 76 69 98 73
RpNR2E6 NR2E6 RPRC009755 b b d d 91 d b b 85 38 88 37
RpDSF NR2E4 RPRC010625 94 70 91 78 d 67 98 81 95 69 86 57
RpSVP NR2F3 RPRC000767 97 d 51 96 d d 97 98 96 98 d 99
RpERR NR3B4 Not annotated 88 c 87 c d c 84 c 87 c 87 c
RpHR38 NR4A4 RPRC001680 96 77 96 82 99 73 96 76 96 79 97 77
RpFTZ-F1 NR5A3 RPRC001915 c c c c c c c c c c c c
RpHR39 NR5B1 RPRC002968 89 80 92 83 93 75 89 87 92 85 94 83
RpHR4 NR6A1 RPRC012796 93 56 99 76 96 61 91 79 93 56 94 d
Note: a, LBD is not present in the NR0 family; b, Gene not present in the species genome; c, Domain truncated in the R. prolixus genome; d, Domain truncated in
the species genome.
R. prolixus nuclear receptor gene annotation 3
© 2021 The Royal Entomological Society, 1–18

two E78 paralogous genes, which originated from a recent
duplication event. However, both E78 genes (named
RpE78-1 and RpE78-2) are highly similar, as shown in
the alignment in Fig. 2. The identity between the proteins’
primary sequence reached 98% within the shared region.
Moreover, an alignment comparing contigs GL561941
and GL558919 (where RpE78-1 and RpE78-2 are, respec-
tively) showed that they are almost identical (99% shared
identity), with GL558919 (the smallest contig) covering
11% of GL561941 (data not shown). This similarity ham-
pers the ability to design specific primers for each
transcript.
The homology analysis for the proteins from the NR2
family also showed a high degree of reliability (Fig. 3(A),
(B)). In the tree generated using the DBD sequences, all
the genes showed clade separation with a monophyletic
origin (Fig. 3(A)). Based on this result, we can conclude
that the R. prolixus genome encodes one orthologue of
each of the following genes: USP,HR78,Tll (Tailless),
HR51,NR2E6,Dsf (Dissatisfaction) and Svp (Seven-up)
(Table 1). Moreover, the kissing bug has two paralogues
of the HNF4 gene (named RpHNF4-1 and RpHNF4-2), a
new duplication within the Insecta class. However, we were
unable to find an orthologue of HR83, even using different
strategies. The NR2 family analysis also showed that the
R. prolixus genome encodes an NR2E6 gene orthologue
(Fig. 3(A), (B)).
The last homology analysis included DBD and LBD from
nuclear receptors within the families NR3, NR4, NR5 and
NR6 (Fig. S4(A), (B)). In the DBD tree, all the families
Figure 1. R. prolixus genome annotation showed a possible E78 gene duplication. The DBD and LBD amino acid sequences from NR1 family proteins in
different species were aligned using ClustalW, and the dendrogram tree was constructed by maximum likelihood method. The bootstrap values are indicated on
branches higher than 50, and the bar indicates the substitutions per site. The first two letters of the protein name indicate the species. Am: Apis mellifera; Ap:
Acyrthosiphon pisum;Bm:Bombyx mori; Dm: Drosophila melanogaster; Dp: Daphnia pulex; Rp: Rhodnius prolixus; and Tc: Tribolium castaneum.
(A) Dendrogram generated with DBD sequences and (B) dendrogram generated with ligand-binding domain sequences.
4 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

constitute monophyletic clades (Fig. S4(A)). As shown in
Fig. 5(B), when we used LBD sequences to generate the
analysis, the NR5 family was separated into a paraphyletic
group but constituted a monophyletic clade when the HR4
gene (from NR6 family) was included (Fig. S4(B)). These
results support the conclusion that the R. prolixus genome
encodes one orthologue for each of the following genes:
FTZ-F1,HR39,HR4,ERR and HR38. Notably, we did not
find the RpERR gene on the genome annotation, and these
data must be included in the next annotation to be
released.
We analysed the expression profiles of the identified
nuclear receptor genes using RT-PCR. For this purpose,
we dissected adult insects on the fourth day after a blood-
meal and obtained their anterior and posterior midguts, fat
body, flight muscle, ovary and testis. Fig. 4 shows the RT-
PCR results for all the genes. The analysed organs broadly
expressed most of the nuclear receptor genes, including
RpKnrl-2,RpE75,RpE78,RpHR3,RpHR96,RpHNF4-2,
RpUSP,RpHR78,RpERR,RpFTZ-F1,RpHR39 and
RpHR4. The other genes had a more restricted expression
profile. RpKnrl-1,RpEcR and RpTLL expressions were
only detected in the fat body and reproductive organs.
Although all the organs expressed RpHNF4-2, the mes-
senger RNA (mRNA) levels of the other paralogue
(RpHNF4-1) were lower in the posterior midgut. All the
organs except the flight muscle expressed RpNR51. The
expression of RpDsf was not detected only in the fat body
and the flight muscle. Finally, RT-PCR analysis only
amplified the RpSpv transcripts in the flight muscle and
ovary and the RpHR38 in the anterior midgut, flight muscle
and reproductive organs. Interestingly, we could not detect
RpNR2E6 expression under the test conditions; however,
this gene may be active in other organs or at a different time
of the feeding cycle.
We then performed a quantitative expression analy-
sis of RpEcR,RpUSP,RpE75,RpHNF4-1 and
RpHNF4-2 by qPCR. We chose to investigate the
expression of these genes due to their role in ecdysone
signalling (RpEcR and RpUSP) or lipid metabolism
(RpE75,RpHNF4-1 and RpHNF4-2). Both the RpEcR
and RpUSP genes were more active in the testicles
than in the midgut and fat body (Fig. 5(A), (B)). The
RpHNF4-1 gene was more highly expressed in the fat
body and ovaries than in the anterior midgut, while
RpHNF4-2 had higher expression in the midgut than in
the other analysed organs. If we compare the expres-
sion of these two paralogues, the RpHNF4-2 gene
was more active in the midgut, testis and flight muscle,
while RpHNF4-1 was more highly expressed in the ova-
ries (Fig. 5(C)). The RpE75 gene, however, showed no
difference in expression between organs (Fig. 5(D)).
We investigated whether feeding could alter the expres-
sion of these genes in the fat body. However, the expres-
sion of all the analysed genes remained stable until
15 days after feeding (Fig. 6). Interestingly, the RpHNF4-2
gene always had higher expression than its RpHNF4-1
paralogue (Fig. 6(C)).
Figure 2. RpE78 paralogues have a high degree of shared identity. The primary sequences of RpE78-1 and RpE78-2 were aligned using ClustalW. (*)
represents identical amino acid; (.) and (:) represent similar amino acid.
R. prolixus nuclear receptor gene annotation 5
© 2021 The Royal Entomological Society, 1–18

Figure 3. R. prolixus has two HNF4 paralogues and one NR2E6 orthologue but has lost the HR83 gene. The DNA-binding domain (DBD) and ligand-binding
domain (LBD) amino acid sequences from NR2 family proteins in different species were aligned using ClustalW, and the dendrogram tree was constructed by
maximum likelihood method. The bootstrap values are indicated on branches higher than 50, and the bar indicates the substitutions per site. The first two letters
of the protein name indicate the species. Am: Apis mellifera; Ap: Acyrthosiphon pisum; Bm: Bombyx mori; Dm: Drosophila melanogaster; Dp: Daphnia pulex;
Rp: Rhodnius prolixus; and Tc: Tribolium castaneum. (A) Dendrogram generated with DBD sequences and (B) dendrogram generated with LBD sequences.
6 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

Next, we explored the effect of 20-HE and juvenile hor-
mone (JH) on lipid metabolism in R. prolixus. Treatment
with 50 ng of 20-HE increased the amount of TAG in the
fat body by approximately 10-fold (Fig. 7). However, JH
had no observable effect on the tested quantities (Fig. S5).
An increase in lipogenesis, a decrease in lipolysis or both
situations could increase the TAG levels. We measured the
acetate incorporation into the fat body to measure the fatty
acid synthesis and explore these hypotheses. Treatment
with 50 ng of 20-HE increased acetate uptake by approxi-
mately 1.5-fold (Fig. 8), indicating hormone-induced lipo-
genesis. However, 20-HE did not induce SREBP
cleavage to its active form (Fig. S6) and did not increase
the ATP-citrate lyase (ACLY) activity (Fig. S7) or alter the
expression of critical genes in the lipid synthesis pathway
(Fig. S8). Thus, the mechanism of action of 20-HE in regu-
lating lipid metabolism in R. prolixus is still unclear and
requires further investigation.
Discussion
First, we identified 22 nuclear receptor superfamily genes
in the R. prolixus genome. In 2016, Vidal and colleagues
investigated the transcription factors present in the
R. prolixus genome and found 19 nuclear receptors. How-
ever, they did not investigate this superfamily in detail
(Vidal et al., 2016). As the authors searched for annotated
genes using conserved domains, they may have missed
the unannotated RpERR gene and paralogues of E78 and
HNF4. The different analysis methods may explain the dif-
ference in the number of genes found. Because we have
also performed an expression analysis of these genes,
we have confidence in our bioinformatics results.
The NR0 proteins’functions are still mostly unknown.
These proteins seem to be involved in the gap gene net-
work during embryonic development in D. melanogaster,
A. gambiae, the moth flyClogmia albipunctata,in
T. castaneum and the milkweed bug Oncopeltus fasciatus
(Nauber et al., 1988; Goltsev et al., 2004; Cerny
et al., 2008; Ben-David and Chipman, 2010; Garcia-
Solache et al., 2010). However, there is no described rela-
tion yet between the NR0 family and metabolic regulation.
Perl and colleagues extensively analysed the evolution of
the NR0 family proteins in R. prolixus. The authors con-
cluded that the two R. prolixus NR0 genes are paralogues,
sharing the knirps-related gene as the same original gene.
Furthermore, the kissing bug (as in A. aegypti) may have
lost the eagle gene orthologue during evolution (Perl
et al., 2013). Based on these results, we named the two
members of the NR0 family RpKnrl-1 (knirps-like) and
RpKnrl-2. In our results, it is possible to support the recent
gene duplication that may have occurred in A. mellifera
(Perl et al., 2013).
Most studied insects have only one E78 gene except for
the fruit flyDrosophila grimshawi, which has two paralo-
gues (Clark et al., 2007). R. prolixus may be the second
case. However, the similarity of the paralogues also
increases the risk of a genomic assembly error. An in-depth
study is needed to solve this question. E78 is an early
Figure 4. R. proxilus organs broadly express most of the nuclear receptor
genes. The organs were obtained from females and males on the fourth day
after a bloodmeal. Total RNA was extracted from the organs and treated
with DNase I. The RNA samples were used for cDNA synthesis, and reverse
transcriptase-PCR (RT-PCR) was performed using specific primers
designed for each gene. RpEF-1 amplification was used as a positive
control. The RT-PCR products were separated for evaluation using agarose
gel electrophoresis. The image shown is representative of three
independent experiments. AM, anterior midgut; PM, posterior midgut; FB,
fat body; FM, flight muscle; OV, ovary; and TE, testis.
R. prolixus nuclear receptor gene annotation 7
© 2021 The Royal Entomological Society, 1–18

inducible gene in the ecdysone signalling cascade (Stone
and Thummel, 1993), but its function remains unknown.
Although metamorphosis and ecdysis regulate this gene,
it is not essential for normal D. melanogaster development
(Russell et al., 1996). However, E78 is vital for establishing
germ stem cells and for the survival of developing ovarian
follicles (Ables et al., 2015). In T. castaneum,E78 is also
involved in reproduction. In the male, this gene regulates
sperm production and its transfer to the female during cop-
ulation (Xu et al., 2012). Moreover, the E78 knockdown
blocks embryo development (Xu et al., 2010). However,
this gene does not seem to be involved in any process per-
taining to energy metabolism regulation.
No insect studied to date has a duplicated HNF4 gene,
even A. pisum, the insect most closely related to
R. prolixus with a sequenced genome. However, we
believe that our dataset and others that were already pub-
lished by other groups sufficiently corroborate the hypothe-
sis that the R. prolixus genome has two HNF4 genes. First,
we partially sequenced the RpHNF4-1 and RpHNF4-2
genes (and also RpEcR and RpUSP), and we obtained
sequences that confirm the presence of these genes in
Figure 5. Testes highly express RpEcR and RpUSP. The organs were obtained from females and males on the fourth day after a bloodmeal. The total RNA was
extracted from the organs and treated with DNase I. RNA samples were used for cDNA synthesis, and gene expression was analysed by quantitative PCR.
RpEF-1 was used as a reference gene. The bars indicate the mean ± SEM. (A) RpEcR expression. The letters indicate significant differences by one-way
ANOVA, followed by Tukey’s posttest, P< 0.05. n≥4. (B) RpUSP expression. The letters indicate significant differences by one-way ANOVA, followed by
Tukey’s posttest, P< 0.05. n≥3. (C) RpE75 expression. P> 0.05 by one-way ANOVA. n≥7. (D) RpHNF4-1 and RpHNF4-2 expression. Lowercase letters
indicate significant differences in RpHNF4-1 expression between different organs by one-way ANOVA, followed by Tukey’s posttest, P< 0.05. Capital letters
indicate significant differences in the expression of RpHNF4-2 between different organs by one-way ANOVA, followed by Tukey’s posttest, P< 0.05. (*), (**) and
(***) indicate a significant difference between the expression of RpHNF4-1 and RpHNF4-2 in each organ, P< 0.05, 0.01 and 0.001, respectively, by unpaired t-
test with a Welch correction. n≥4.
8 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

the genome (data not shown). Second, the qPCR
results corroborate that they are different genes. The
expression profiles of RpHNF4-1 and RpHNF4-2 are
significantly different between the organs and, over
the time course, in the fat body. The only differences
in the amplification efficiency of the primers are unable
to explain these results. Third, we also used RNA inter-
ference, uncovering data that we will publish in another
manuscript. We used two different primer pairs for each
gene to produce templates for double-stranded RNA
(dsRNA) production, and in all cases, the amplification
products were of the expected size. In addition, the
obtained gene knockdown was specific for each paralo-
gue (data not shown). Fourth, published transcriptome
data confirm the two RpHNF4 paralogues (Ribeiro
et al., 2014; Ons et al., 2016; Latorre-Estivalis
et al., 2020). Finally, although we have not sequenced
the qPCR amplification products, the products have
the predicted melting temperature values in the qPCR
dissociation curve based on the amplicon sequence.
Figure 7. 20-Hydroxyecdysone induces triacylglycerol (TAG) accumulation
in the fat body. Fasting females were injected with different amounts of
20-HE and then fed. Two days after treatment, the insects were dissected,
and their fat bodies were obtained. The amount of TAG in the samples was
measured by colorimetric assay. The bars indicate the means ± SEM. (**)
indicates a significant difference from the control values by one-way
ANOVA, followed by the Holm-Sidak’s multiple comparison posttest,
P< 0.01. n ≥7.
Figure 6. Feeding does not regulate the expression of RpEcR,RpUSP,RpE75,RpHNF4-1 and RpHNF4-2 in the fat body. Fat bodies were obtained from
fasting females or at different days after feeding. The total RNA was extracted from the organs and treated with DNase I. The RNA samples were used for cDNA
synthesis, and the gene expression was analysed by quantitative PCR. RpEF-1 was used as a reference gene. The symbols represent the mean ± SEM.
RpEcR (A) and RpUSP (B) expression. P> 0.05 by one-way ANOVA. n≥4. (C) RpE75 expression. P> 0.05 by one-way ANOVA. n≥3. (D) RpHNF4-1 and
RpHNF4-2 expression. (*), (**) and (***) indicate a significant difference between the expression of RpHNF4-1 and RpHNF4-2 on each day, P< 0.05, 0.01 and
0.001, by two-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons posttest. n=6.
R. prolixus nuclear receptor gene annotation 9
© 2021 The Royal Entomological Society, 1–18

Thus, we guarantee that the primers amplify RpHNF4-1
and RpHNF4-2 specifically.
The HNF4 gene experienced a vast expansion within the
nematodes, and the genome of the worm Caenorhabditis
elegans encodes more than 270 nuclear receptors, primar-
ily due to multiples HNF4 duplications (Robinson-Rechavi
et al., 2005). In insects, HNF4 is activated by fatty acid
binding (Palanker et al., 2009), although this protein also
binds to heme with a low affinity (De Rosny et al., 2008).
The primary organs involved in metabolic control in
D. melanogaster express this gene, and dhnf4 knockout
flies exhibit a variety of phenotypes that include starvation
sensitivity, low TAG storage mobilization, long-chain fatty
acid accumulation and the inhibited expression of genes
involved in lipolysis and β-oxidation (Palanker
et al., 2009). These phenotypes led to the hypothesis that
insect HNF4 is a functional analogue of the mammalian
peroxisome proliferator-activated receptor αnuclear recep-
tor (Palanker et al., 2009). HNF4 knockdown in A. aegypti
yields similar results; mosquitoes show reduced TAG
catabolism and β-oxidation enzymes and increased TAG
levels. Importantly, HNF4 exerts this regulation directly by
binding to the gene promoter (Wang et al., 2017). HNF4
knockout D. melanogaster flies also demonstrate changes
in carbohydrate metabolism, increased glycaemia and glu-
cose intolerance and reduced insulin secretion, presenting
characteristics of diabetes models (Barry and
Thummel, 2016). HNF4 is also involved in synthesizing
the very long-chain fatty acids and hydrocarbons neces-
sary for protection against desiccation in adult flies
(Storelli et al., 2019). T. castaneum reproduction involves
HNF4, for example, E78.HNF4 knockdown reduces male
fertility (Xu et al., 2012) and eggs hatching
(Xu et al., 2010). In that context, the HNF4 duplication in
R. prolixus become an interesting point of investigation.
Understanding if both paralogues have similar and redun-
dant functions or assumed different roles in metabolism
control will lead to new data about the evolution of the
nuclear receptor superfamily and its relation to energy
metabolism. Our results show that the two R. prolixus
HNF4 genes have different expression profiles, indicating
that they have different metabolic functions or specialize
in acting on specific organs. Functional genetic analyses
are necessary to investigate these hypotheses.
The NR2 family analysis also showed that the R. prolixus
genome encodes an NR2E6 gene orthologue. This gene is
absent in D. melanogaster and D. pseudoobscura and in
A. pisum, but it can be found in the genomes of
A. gambiae,A. aegypti,B. mori,T. castaneum,
A. mellifera and N. vitripennis (Fahrbach et al., 2012). In
their review article, Fahrbach and colleagues speculated
that NR2E6 should be an ancient gene, lost in the Drosoph-
ila lineage and in A. pisum. They also predicted that other
hemimetabolous genomes would contain the NR2E6 gene,
and the R. prolixus genome confirmed that idea. Although
the genome data are still scarce, we can conclude by now
that the ancestral Hemiptera should have an NR2E6 gene
and that the Aphididae lineage may have lost it, but the
Reduviidae lineage retained the gene. The function and
ligand of NR2E6 are unknown, making it impossible to
speculate about the impact of having or not having this
gene on the insect’s metabolism.
R. prolixus does not have an HR83 gene orthologue.
This gene is present in most studied insects, except for
B. mori (Fahrbach et al., 2012). This result indicates that,
as in NR2E6,HR83 may have been lost several times inde-
pendently during insect evolution. However, analyses of
other insect genomes are necessary to trace when these
events occurred. As in NR2E6, the HR83 function is
unknown, and an orthologous gene is absent in mammals
(Weber et al., 2012). Nevertheless, although we have used
different strategies to locate the gene, we cannot exclude
the possibility that HR83 is present in the R. prolixus
genome and that someone may find it during their next
annotation efforts.
We analysed the gene expression profile of nuclear
receptors identified in the different organs of R. prolixus.
The analysed organs expressed the most genes. These
expression profiles were similar to those published for
A. aegypti (Cruz et al., 2009), except that we could not
detect RpNR2E6 expression, and RpSvp demonstrated
activity only in the ovary but not in the fat body.
A quantitative analysis showed that the testis is the
organ with higher expression of RpEcR and RpUSP.In
the cockroach Periplaneta americana, USP is more highly
expressed in the fat body, followed by the ovaries and mus-
cle, but the authors did not analyse the testis (Elgendy
Figure 8. 20-Hydroxyecdysone (20-HE) induces de novo fatty acids
synthesis in the fat body. Fasting females were injected with 50 ng of 20-HE
and then fed. Two days after the treatment, the insects were dissected, and
the fat body was left attached to the ventral cuticle. The organs were
incubated with a culture medium containing radio-labelled acetate, and after
1 h, the fat body lipids were extracted. The radioactivity present in the
extracted lipids was used to estimate the incorporation of acetate. The bars
indicate the means ± SEM. (*) indicates a significant difference by unpaired
t-test, P< 0.05. n ≥9.
10 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

et al., 2014). None of the analysed genes showed signifi-
cant expression changes in the fat body after feeding by
R. prolixus. Due to the increase in 20-HE levels in the hae-
molymph after feeding (Cardinal-Aucoin et al., 2013), we
expected to observe an increase in the expression of
genes regulated by this hormone, such as E75, as already
shown in A. aegypti (Pierceall et al., 1999). In lepidop-
terans, EcR,USP and E75 gene activity increased during
the larva–pupa transition period, following the circulating
20-HE levels (Huang et al., 2015; Li et al., 2015; Peng
et al., 2018). The nymphs of the cockroach Blatella germa-
nica have a similar expression pattern for the E75 gene,
which has increased activity before the change between
stages (Mané-Padrós et al., 2008). In P. americana adults,
the USP gene showed increased expression in the fat body
5 and 9 days after adult emergence, along with increased
vitellogenesis (Elgendy et al., 2014). The absence of gene
expression induction in the fat body may indicate some
type of change only in terms of protein or posttranslational
modifications. However, we have not found studies corre-
lating the levels of mRNA and protein for these nuclear
receptors.
The amount of TAG in the fat body of R. prolixus
increases after feeding, reaching its maximum within
5 days (Pontes et al., 2008). Considering that the 20-HE
levels in the haemolymph reach a peak 4 days after feed-
ing (Cardinal-Aucoin et al., 2013), our result indicating that
20-HE induces lipogenesis in the fat body is attractive.
However, this result is different from the previously
described effects of 20-HE on fat body lipid metabolism in
other insects. In D. melanogaster larvae and A. aegypti
adults, disrupting 20-HE signalling by EcR knockdown
increases the TAG stores in the fat body (Kamoshida
et al., 2012; Wang et al., 2017). In mosquitoes, reduced
TAG catabolism and β-oxidation appear to be responsible
for this effect (Wang et al., 2017). An increase in lipolytic
activity also occurs in B. mori larvae treated with 20-HE
(Wang et al., 2010). However, in adult D. melanogaster,
treating with 20-HE increases the TAG reserves due to
increased food consumption (Sieber and
Spradling, 2015). Thus, it is essential to highlight that the
effects of 20-HE on lipid metabolism may differ between
hemimetabolic and holometabolic insects due to the role
of the hormone in metamorphosis or at different stages of
development. For example, in the planthopper Nilaparvata
lugens, a hemipteran insect such as R. prolixus, 20-HE
increases the lipophorin receptor expression, which is
essential for lipid accumulation in the insect
(Lu et al., 2018). However, the authors did not directly mea-
sure the effect of the 20-HE treatment on the TAG levels.
Thus, we are the first to describe the ability of 20-HE to
induce lipogenesis.
The mechanism by which 20-HE increases lipogenesis
in R. prolixus is not yet clear. We tested different
hypotheses but could not understand how 20-HE stimu-
lates acetate incorporation. In adult D. melanogaster, the
disruption of 20-HE signalling reduces the expression and
activation of SREBP, a central lipogenesis activator
(Sieber and Spradling, 2015). However, in R. prolixus,
treatment with 20-HE did not alter the expression or activa-
tion of SREBP. Similarly, 20-HE did not induce ACLY activ-
ity or the expression of various lipogenic genes. Other
hypotheses could explain how 20-HE increases lipid syn-
thesis. For example, in D. melanogaster, 20-HE increases
the cytoplasmic malic enzyme (cME) activity, which is cru-
cial for de novo fatty acid synthesis (Farkas et al., 2002). In
R. prolixus, 20-HE did not alter the expression of cME, but
we did not measure its activity. Changes in the acetyl-CoA
carboxylase, fatty acid synthase or amino acid transami-
nases activities could also be responsible for increasing
the lipid synthesis, but we still need to test these hypothe-
ses. We chose to perform biochemical tests 2 days after
feeding because we believe that, during this period, the
fat body has more significant lipogenic activity based on
the described TAG dynamics (Pontes et al., 2008). In addi-
tion, the amount of ecdysone in the haemolymph increases
after feeding, reaching a peak on the fourth day after feed-
ing (Cardinal-Aucoin et al., 2013). Therefore, we avoid a
more significant influence from the endogenous ecdysone
by performing the experiments before that peak.
We also tested the effect of JH on the accumulation of
TAG in the fat body. Several previous results have
described that this hormone acts on lipid metabolism in
insects, such as mosquitoes (Sim and Denlinger, 2013;
Hou et al., 2015), bees (Paes-De-Oliveira et al., 2013) and
crickets (Zera and Zhao, 2004). However, in R. prolixus,
JH has no observable effect on the fat body’s TAG amount.
In summary, we have shown that the R. prolixus genome
encodes 22 nuclear receptors and has a duplication of the
HNF4 gene that has never been observed before in insects.
The analysed organs expressed most of these genes. In
addition, 20-HE increases the TAG stores in the fat body
by inducing fatty acid synthesis. However, the details on
the mechanism of this endocrine action are still unclear.
Experimental procedures
Ethical statement
All the animal care and experimental protocols were conducted fol-
lowing the guidelines of the institutional animal care and use com-
mittee (Committee for Evaluation of Animal Use for Research at
the Universidade Federal do Rio de Janeiro, CEUA-UFRJ) and
the National Institutes of Health Guide for the Care and Use of Lab-
oratory Animals (ISBN 0-309-05377-3). CEUA-UFRJ approved
the protocols. The technicians at the animal facility at the Instituto
de Bioquímica Médica Leopoldo de Meis (UFRJ) conducted all
aspects of rabbit husbandry under strict guidelines to ensure the
careful and consistent handling of the animals.
R. prolixus nuclear receptor gene annotation 11
© 2021 The Royal Entomological Society, 1–18

Nuclear receptors genes annotation
The nuclear receptor genes from D. melanogaster were obtained
from the FlyBase database (McQuilton et al., 2012) based on the
gene annotations published elsewhere (King-Jones and
Thummel, 2005). These genes were used as a query in a search
against the R. prolixus preliminary gene annotation from Vector-
Base (Megy et al., 2012) through the BlastP algorithm (Altschul
et al., 1997). The sequences obtained from BlastP were then ana-
lysed using the conserved domain database (CDD) (Marchler-
Bauer et al., 2015) to identify the DBD and LBD domains. If a
nuclear receptor gene was not found in the preliminary annotation,
the DBD and LBD consensus sequences were obtained from the
CDD and used as a query against the R. prolixus genome contigs
sequences through the TBlastN algorithm (Altschul et al., 1997).
When the DBD or the LBD from the R. prolixus gene was found
to be truncated, the respective contig sequence was reanalysed
using the GeneWise algorithm (McWilliam et al., 2013) and the
protein sequence from A. pisum,A. mellifera,D. melanogaster,
T. castaneum,B. mori and A. aegypti (King-Jones and
Thummel, 2005; Velarde et al., 2006; Bonneton et al., 2008; Cheng
et al., 2008; Cruz et al., 2009; Christiaens et al., 2010) to try to cor-
rect the preliminary annotation.
Homology analysis
Only the DBD and LBD sequences obtained from the CDD were used
in the homology analysis. The nuclear receptor domains from
R. prolixus,A. pisum,A. mellifera,D. melanogaster,T. castaneum,
B. mori,A. aegypti and the water flea Daphnia pulex (Thomson
et al., 2009) were aligned using ClustalW 2.0 (Larkin et al., 2007).
The dendrograms were constructed using the maximum likelihood
method (Felsenstein, 1981) with 500 bootstrap replications in MEGA
6.0 software (Tamura et al., 2013). Bootstrap values of less than
50 have been suppressed for easy viewing of the dendrograms.
The D. pulex sequences were used as an outgroup.
Gene expression analysis
The insects (five insects per time point) were dissected after fast-
ing or on the 2nd, 4th, 7th and 15th days after feeding. The anterior
and posterior midgut, fat body, ovary, testis and flight muscle were
collected. The obtained organs were washed in 0.15 M NaCl and
homogenized in 500 μl of RNA extraction solution (phenol 38%
v/v, 1 M guanidinium thiocyanate, 1 M ammonium thiocyanate,
100 mM sodium acetate and glycerol 5% v/v; Sigma-Aldrich),
and the total RNA was extracted as described elsewhere
(Rodríguez-Ezpeleta et al., 2009). The total RNA concentrations
were determined using a NanoDrop Lite Spectrophotometer
(Thermo Fisher Scientific, Waltham, MA, USA), and all the RNA
samples included in the further analysis had an absorbance at
260 nm/absorbance at 280 mm (A260/A280) ratio between 1.8
and 2.0. RNA integrity was checked using native agarose gel elec-
trophoresis. The RNA samples were considered intact when an
18S band was observed. The band corresponding to 28S rRNA
could not be identified because of the ’hidden break’present in
the insects (Ishikawa, 1977; Winnebeck et al., 2010). A 1 μg sam-
ple of RNA was treated with 1 U of RNase-free DNase I (Sigma-
Aldrich) for 30 min at 37 Cinafinal volume of 10 μl. The DNAse
I reaction was stopped by adding 50 nmol of
ethylenediaminetetraacetic acid and incubating at 65 C for
10 min. The treated RNA was then used to synthesize cDNA sam-
ples using a High Capacity cDNA Reverse Transcription Kit
(Thermo Fisher Scientific) in a final reaction volume of 22 μl. Each
reaction mixture contained 55 U of MultiScribe™MuLV reverse
transcriptase, and cDNA synthesis was performed with random
primers. The reactions were incubated at 37 C for 2 h. Control
reactions without reverse transcriptase were performed to confirm
the efficiency of the DNaseI treatment.
For RT-PCR, the reactions were performed using PCR Master
Mix (Promega, Fitchburg, WI, USA) and 3.0 pmol of each primer
(Exxtend Biotecnologia, Paulínia, Brazil). The primers used for
each gene were designed using the Primer3 algorithm (Rozen
and Skaletsky, 2000) and are shown in Table S1. The temperature
variation programme included the following steps: 94 C for 2 min,
40 cycles of 94 C for 30 s, 60 C for 30 s and 72 C for 30 s fol-
lowed by 72 C for 10 min. RpEF-1 gene amplification was used
as a positive control. The PCR products were then visualized using
native agarose gel electrophoresis.
The qPCR reaction was performed in a StepOnePlus thermocy-
cler (Thermo Fisher Scientific). The reaction mixture contained
10-fold diluted JumpStart Taq DNA polymerase buffer (Sigma-
Aldrich), 5 mM MgCl
2
(Sigma-Aldrich), 20 000-fold diluted SYBR
Green I (Sigma-Aldrich), 0.2 mM deoxynucleotide triphosphate
(Thermo Fisher Scientific), 0.375 U of JumpStart Taq DNA poly-
merase (Sigma-Aldrich), 100-fold diluted reference dye (Sigma-
Aldrich), forward and reverse primers as described in Table S2
and 0.3 μl of the cDNA sample in a final volume of 15 μl. This reac-
tion mixture was based on descriptions published elsewhere
(Karsai et al., 2002). The primers were designed using the Primer3
algorithm (Rozen and Skaletsky, 2000) and are described in
Table S2. The amplification efficiency was determined according
to the cycles of quantification (Cq) obtained from qPCR using
cDNA that was serially diluted as a template. The amplification effi-
ciency, cDNA curve slope, intercept yand r
2
are described in
Table S2. The primers used for RpACC,RpACSL-1,RpDGAT
and RpGPAT amplification were described elsewhere (Alves-
Bezerra and Gondim, 2012; Alves-Bezerra et al., 2016b; Majero-
wicz et al., 2016). For blank controls, the cDNA was replaced with
nuclease-free water. The Cq values obtained for the blank controls
were at least 10 units higher than those obtained for the experi-
mental points. The Cq values obtained for the cDNA controls syn-
thesized without reverse transcriptase were at most five units
lower than those obtained for the blank controls. The reactions
were performed in MicroAmp Fast Optical 96-well plates (Thermo
Fischer Scientific) under the following conditions: 10 min at 95 C
followed by 40 cycles of 15 s at 95 C and 1 min at 60 C and a dis-
sociation curve. The RpEF-1 gene expression was used as a refer-
ence (Majerowicz et al., 2011). The ΔCq values were calculated
using the obtained Cq values, as described in the literature
(Livak and Schmittgen, 2001). These values were used for statisti-
cal analyses. The relative expression values (2
−ΔCq
) were used
only for plotting the graphs. The gene expression values are given
relative to the expression of the reference gene.
Treatment with 20-HE and JH (JH-III)
For treatment with 20-HE (Sigma-Aldrich), fasting females were
injected with increasing amounts (5 pg, 50 pg, 500 pg, 5 ng and
12 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18

50 ng) of the hormone diluted in phosphate-buffered saline (PBS,
10 mM phosphate buffer; pH 7.4, 150 mM NaCl) containing 2%
dimethyl sulfoxide (DMSO) v/v. The insects were injected with a
10-μl syringe (Hamilton Company, Reno, NV, USA).
For JH-III (Sigma-Aldrich) treatment, fasting females were
exposed in the ventral region to increasing amounts (3 pg, 30 pg,
300 pg, 3 ng and 30 ng) of the hormone diluted in acetone.
In both cases, the insects were fed immediately after the treat-
ments. In these experiments, only females were used as males
do not have the TAG dynamics in the fat body, as described else-
where (Pontes et al., 2008).
TAG quantification
The amount of TAG stored in the animals was measured using the
enzymatic colorimetric assay Triglicérides (Interteck Katal, Belo
Horizonte, Brazil), as described elsewhere (Alves-Bezerra
et al., 2016a). In brief, 2 days after treatment, the insects were dis-
sected, and the resulting fat bodies were individually homogenized
in 100 μl of PBS in a 1.5-ml microcentrifuge tube using a plastic
pestle. The homogenate was used immediately or frozen at −20
C without undergoing thermal inactivation. The samples (10 μl)
were mixed with 200 μl of the colour reagent and incubated at 37
C for 30 min. The incubations were performed in 96-well plates.
The absorbance was measured at 540 nm, and the amount of
TAG was calculated by comparing the absorbance to that of a
standard glycerol curve.
The amount of total protein, as measured by Lowry’s method
(Lowry et al., 1951), was used to normalize the sample results. In
brief, the samples (0.5 μl) were added to 200 μl of Lowry’s solution
and 20 μl of the Folin–Ciocalteu reagent (Sigma-Aldrich). The
incubations were performed in 96-well plates. The absorbance
was measured at 660 nm, and the protein amount was calculated
by comparing the absorbance to that of a standard bovine serum
albumin (Sigma-Aldrich) curve.
De novo lipid synthesis assay by ex vivo acetate
incorporation
The de novo lipid synthesis assay was performed as previously
described elsewhere (Alves-Bezerra et al., 2016b). In brief, the
insects were dissected 2 days after treatment, and the internal
organs were removed. The fat body was kept in association with
the ventral cuticle and incubated ex vivo in a humid chamber with
20 μl of medium 199 (Sigma-Aldrich) supplemented with sodium
phosphate (10 mM, pH 7.4) and sodium acetate (10 mM) contain-
ing 0.1 μCi radioactive
3
H-acetate (Perkin-Elmer, Waltham, MA,
USA) for 1 h at room temperature. Following the incubation, the
organs were washed, dissected and homogenized in 800 μlof
PBS. The lipids were extracted (Blight and Dyer, 1959), and ace-
tate uptake was estimated by the presence of radioactivity in the
organic phase as measured by liquid scintillation in a Tri-Carb
2800TR scintillator (Perkin-Elmer).
Western blot
The insects were dissected 2 days after treatment, and the fat bod-
ies were individually homogenized in 50 μl of buffer (5 mM Tris–
HCl; pH 7.4, 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM
NaF, 2 mM VO
43−
and protease inhibitors (Sigma-Aldrich)). The
amount of protein was dosed as described above. The samples
(40 μg) were separated on 10% sodium dodecyl sulphate poly-
acrylamide gel electrophoresis and transferred to a polyvinylidene
fluoride (PVDF) membrane. The membrane was blocked with
TBSTM (20 mM Tris–HCl; pH 7.5, 150 mM NaCl, 0.1% v/v
Tween-20, 5% w/v skim milk) for 1 h at room temperature and incu-
bated with anti-SREBP N-terminal region antibody (Santa Cruz
Biotechnology, Dallas, TX, USA) at 1:1000 v/v dilution in TBSTM
for 16 h at 4 C. The membrane was then washed and incubated
with peroxidase-coupled rabbit anti-IgG antibody (Santa Cruz) at
a 1:10 000 v/v dilution in TBSTM for 1 h at room temperature.
The membrane was washed, incubated with Na ECL Prime West-
ern Blotting System (Sigma-Aldrich) and scanned on ChemiDoc
XRS+ (Bio-Rad Laboratories, Hercules, CA, USA).
ACLY activity assay
The insects were dissected 2 days after treatment, and the fat bod-
ies were homogenized in 0.1 mM Tris–HCl buffer, at a pH of 7.2.
The samples were centrifuged at 1000gfor 10 min at 4 C, and
the supernatant was collected. The amount of protein was dosed
as described above. The reactions contained 100 mM Tris–HCl,
pH 7.3, 10 mM MgCl2, 20 mM citrate, 5 mM ATP, 10 mM
2-mercaptoethanol, 2 U/ml malate dehydrogenase, 0.15 mM
NADH, 0.3 mM coenzyme A and 20 μg of protein. The blanks reac-
tions did not contain coenzyme A. The reactions were incubated at
37 C for 1 h, with absorbance readings at 340 mM every minute.
The ACLY activity was estimated by the absorbance reduction at
340 nm, corresponding to NADH oxidation.
Acknowledgements
We thank Dr. Hatisaburo Masuda for providing the
R. prolixus. We also thank José de S. Lima Junior, Gustavo
Ali, Desenir A. Pedro and Yasmin P. Gutierrez for insect
care. This work was supported by grants from the
Fundaç~
ao de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ), the Coordenaç~
ao de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES) and the Conselho
Nacional de Pesquisa e Desenvolvimento (CNPq).
Data availability statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
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Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the
article.
Figure S1: General homology analysis using nuclear receptor DBD
sequences.
The amino acid sequences of the nuclear receptor DBDs from different spe-
cies were aligned using ClustalW, and the dendrogram tree was con-
structed by neighbour-joining method. The bootstrap values are indicated
on branches only when higher than 50, and the bar indicates the substitu-
tions per site. The first two letters of the protein name indicate the species.
Am: Apis mellifera; Ap: Acyrthosiphon pisum; Bm: Bombyx mori; Dm: Dro-
sophila melanogaster; and Tc: Tribolium castaneum;R. prolixus genes are
denoted by their annotation codes in VectorBase. “Trunk”means the
domain sequence is not complete upon annotation.
Figure S2: General homology analysis using nuclear receptor LBD
sequences.
The amino acid sequences of the nuclear receptor LBDs from the different
species were aligned using ClustalW, and the dendrogram tree was con-
structed by neighbour-joining method. The bootstrap values are indicated
on branches only when higher than 50, and the bar indicates the substitu-
tions per site. The first two letters of the protein name indicate the species.
Am: Apis mellifera; Ap: Acyrthosiphon pisum; Bm: Bombyx mori; Dm: Dro-
sophila melanogaster; and Tc: Tribolium castaneum;R. prolixus genes are
denoted by their annotation codes in VectorBase. “Trunk”means that the
domain sequence is not complete at annotation.
Figure S3:R. prolixus genome encodes two NR0 family genes.
The DBD amino acid sequences from the NR0 family proteins from different
species were aligned using ClustalW, and the dendrogram tree was con-
structed by maximum likelihood method. The bootstrap values are indicated
on branches higher than 50, and the bar indicates the substitutions per site.
The first two letters of the protein name indicate the species. Am: Apis mel-
lifera; Ap: Acyrthosiphon pisum;Bm:Bombyx mori; Dm: Drosophila mela-
nogaster; Dp: Daphnia pulex; Rp: Rhodnius prolixus; and Tc: Tribolium
castaneum.
Figure S4:R. prolixus has one-to-one orthologues within NR3, NR4, NR5
and NR6 families.
The DBD and LBD amino acid sequences from proteins of the NR3, NR4,
NR5 and NR6 families in different species were aligned using ClustalW,
and a dendrogram tree was constructed by maximum likelihood method.
The bootstrap values are indicated on branches higher than 50, and the
bar indicates the substitutions per site. The first two letters of the protein
name indicate the species. Am: Apis mellifera; Ap: Acyrthosiphon pisum;
Bm: Bombyx mori; Dm: Drosophila melanogaster; Dp: Daphnia pulex; Rp:
Rhodnius prolixus; and Tc: Tribolium castaneum. (A): Dendrogram gener-
ated with DBD sequences and (B): dendrogram generated with LBD
sequences.
Figure S5: JH does not induce TAG accumulation in the fat body.
Fasting females were treated with different amounts of JH-III and then fed.
Two days after treatment, the insects were dissected, and their fat bodies
were obtained. The amount of TAG in the samples was measured by color-
imetric assay. Bars indicate means ± SEM. P> 0.05 by one-way
ANOVA. n≥9.
Figure S6: 20-HE does not activate SREBP in the fat body.
Fasting females were injected with 50 ng of 20-HE and then fed. Two days
after treatment, the insects were dissected, and their fat bodies were
obtained. (A) The protein samples were analysed by Western blot using
the anti-N-terminal SREBP region antibody. The bands were developed
using a luminescence scanner. A representative image of three experi-
ments. (B) The intensity of the precursor and cleaved SREBP bands were
analysed by densitometry. Bars indicate means ± SEM. P= 0.1255 by
Mann–Whitney test. n≥5.
Figure S7: 20-HE does not regulate ACLY activity in the fat body.
Fasting females were injected with 50 ng of 20-HE and then fed. Two days
after treatment, the insects were dissected, and the fat bodies were
obtained. The ACLY activity was measured by NAD reduction-coupled
enzymatic method. Bars indicate means ± SEM. P= 0.5592 by unpaired t-
test. n=6.
Figure S8: 20-HE does not regulate the expression of genes involved in
lipogenic pathways. Fasting females were injected with 50 ng of 20-HE
and then fed. One (A) or two days (B) after treatment, the insects were dis-
sected, and the total RNA was extracted from the collected fat bodies. The
RNA samples were treated with DNAseI and used for cDNA synthesis.
The gene expression was analysed by qPCR, and the RpEF-1 gene was
used as a reference gene. The bars indicate the means ± SEM. P> 0.05
by unpaired t-test. n≥3. ACLY: ATP citrate lyase; ACC: acetyl-CoA carbox-
ylase; FAS: fatty acid synthase; ACSL-1: acyl-CoA synthetase long-chain;
GPAT: glycerol-3-phosphate acyltransferase; DGAT: diacylglycerol acyl-
transferase; G6PDH: glucose-6-phosphate dehydrogenase; cME: cytoplas-
mic malic enzyme; and SREBP: sterol-responsive element-binding protein.
Table S1: Sequences of primers used for RT-PCR in this work.
Table S2: Sequences, concentrations and information obtained from cali-
bration curves of primers used in qPCR.
18 P. V. P. Nascimento et al.
© 2021 The Royal Entomological Society, 1–18